The invention relates generally to immunoconjugates for diagnostic and therapeutic uses in cancer. In particular, the invention relates to recombinantly produced humanized monoclonal antibodies directed against B-cell lymphoma and leukemia cells, which antibodies can be covalently conjugated to a diagnostic or therapeutic reagent without loss of antibody binding and internalization function and with reduced production of human anti-mouse antibodies.
Non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia are B-cell malignancies that remain important contributors to cancer mortality. The response of these malignancies to various forms of treatment is mixed. They respond reasonably well to chemotherapy, and, in cases where adequate clinical staging of NHL is possible, as for patients with localized disease, satisfactory treatment may be provided using field radiation therapy (Hall et al., Radiology for the Radiologist, Lippincott, Philadelphia, 1989, pp 365-376). However, the toxic side effects associated with chemotherapy and the toxicity to the hematopoietic system from local, as well as whole body, radiotherapy, limits the use of these therapeutic methods. About one-half of the patients die from the disease (Posner et al., Blood, 61: 705 (1983)).
The use of targeting monoclonal antibodies conjugated to radionuclides or other cytotoxic agents offers the possibility of delivering such agents directly to the tumor site, thereby limiting the exposure of normal tissues to toxic agents (Goldenberg, Semin. Nucl. Med., 19: 332 (1989)). In recent years, the potential of antibody-based therapy and its accuracy in the localization of tumor-associated antigens have been demonstrated both in the laboratory and clinical studies (see., e.g., Thorpe, TIBTECH, 11: 42 (1993); Goldenberg, Scientific American, Science & Medicine, 1: 64 (1994); Baldwin et al., U.S. Pat. Nos. 4,925,922 and 4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095; Irie et al., U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat. Nos. 5,134,075 and 5,171,665). In general, the use of radio-labeled antibodies or antibody fragments against tumor-associated markers for localization of tumors has been more successful than for therapy, in part because antibody uptake by the tumor is generally low, ranging from only 0.01% to 0.001% of the total dose injected (Vaughan et al., Brit. J. Radiol., 60: 567 (1987)). Increasing the concentration of the radiolabel to increase the dosage to the tumor is counterproductive generally as this also increases exposure of healthy tissue to radioactivity.
LL2 (EPB2) is a highly specific anti-B-cell lymphoma and anti-lymphocytic leukemia cell murine monoclonal antibody (mAb) that is rapidly internalized by such cells and that can overcome some of the aforementioned difficulties (Shih et al., Int. J. Cancer, 56: 538 (1994)). LL2, which is of the IgG2a antibody type, was developed using the Raji B-lymphoma cell line as the source of antigen (Pawlak-Byczkowska et al., Cancer Res., 49: 4568 (1989)). Murine LL2 (mLL2) is known to react with an epitope of CD22 (Belisle et al., Proc Amer. Assn. Clin. Res., 34: A2873 (1993)). CD22 molecules are expressed in the cytoplasm of progenitor and early pre-B cells, and appear in the cell surface of mature B-cells.
By immunostaining of tissue sections, mLL2 was shown to react with 50 of 51 B-cell lymphomas tested. mLL2 provides a highly sensitive means of detecting B-cell lymphoma cell in vivo, as determined by a radioimmunodetection method (Murthy et al., Eur. J. Nucl. Med., 19: 394 (1992)). The Fab' fragment of mLL2 labeled with .sup.99m Tc localized to 63 of 65 known lesions in Phase II trial patients with B-cell lymphoma (Mills et al., Proc. Amer. Assn. Cancer Res., 14: A2857 (1993)). In addition, .sup.131 I-labeled mLL2 was therapeutically effective in B-cell lymphoma patients (Goldenberg et al., J. Clin. Oncol., 9: 548 (1991)). mLL2 Fab' conjugated to the exotoxin PE38KDEL induced complete remission of measurable human lymphoma xenografts (CA-46) growing in nude mice (Kreitman et al., Cancer Res., 53: 819 (1993)).
The clinical use of mLL2, just as with most other promising murine antibodies, has been limited by the development in humans of a human anti mouse antibody response (HAMA). While a HAMA response is not invariably observed following injection of mLL2, in a significant number of cases patients developed HAMA following a single treatment with mLL2. This can limit the diagnostic and therapeutic usefulness of such antibody conjugates, not only because of the potential anaphylactic problem, but also as a major portion of the circulating conjugate may be complexed to and sequestered by the circulating anti-mouse antibodies. This is exemplified by one study in which about 30% of the patients developed low level HAMA response following a single injection of about 6 mg of mLL2 .sup.131 I-IgG and nearly all developed a strong HAMA response with additional injections. On the other hand, with mLL2 Fab' labeled with .sup.99m Tc, no HAMA response was observed. Such HAMA responses in general pose a potential obstacle to realizing the full diagnostic and therapeutic potential of the mLL2 antibody.
As noted above, the use of fragments of mLL2, such as F(ab').sub.2 and Fab', partially alleviates/circumvents these problems of immunogenicity. However, there are circumstances in which whole IgG is more desirable, such as when induction of cellular immunity is intended for therapy, or where an antibody with enhanced survival time is required.
For monoclonal antibodies to function as the delivery vehicles for drugs and radionuclides, it is of prime importance to develop methods for their site-specific conjugations, with minimal perturbation of the resultant immunoreactivities. Most commonly, the conjugation of drugs and radionuclides are accomplished through their covalent attachments to side chains of amino acid residues. Due to the non-site-restricted nature of these residues, it is difficult to avoid undesirable couplings at residues that lie within or are in close vicinity to the ABS, leading to reduced affinity and heterogenous antigen-binding properties. Alternatively, conjugation can be directed at sulfhydryl groups. However, direct labeling relies on the reduction of S-S bonds, with the possible risk of protein fragmentation.
U.S. patent application Ser. No. 08/289,576, now abandoned, but refiled as continuation application, U.S. patent application Ser. No. 08/690,102, now U.S. Pat. No. 5,789,554, issued on Aug. 4, 1998, the entire disclosure of which is incorporated herein by reference, discloses a humanized mAb having a naturally occurring N-linked glycosylation site found at amino acid positions 18-20 of the LL2 VK domain for site-specific drug or chelate conjugation. The attached carbohydrate moiety was positioned away from, and demonstrated no physical contacts with, the antigen binding site (ABS). The immunoreactivity of the antibody was not affected when chelates such as DTPA were conjugated to the carbohydrate.
However, there are limitations to the usefulness of this antibody. For one, it is not clear what size and type of chelates can be attached before immunoreactivity is affected. We have determined that attachment of larger chelates does affect the binding affinity. Thus, attachment of an 18 kD Dox-dextran to the carbohydrate at position 18-20 of the LL2 VK domain reduces immunoreactivity to about 50%. Furthermore, it would be very advantageous to engineer other antibodies to contain active glycosylation sites. Engineering other antibodies so that glycosylation sequences are present in the variable region is difficult because the engineering steps would need to be repeated for each antibody. Furthermore, the immunoreactivity of the construct might be affected.
IgG glycosylation at Asn-297 in the CH2 Fc domain has been well-characterized as important for the maintenance of antibody stability and the appropriate structure for proper effector functions. See Tao and Morrison, J. immunol. 143: 2595 (1989). Due to the restricted localization of immunoglobulin glycosylation sites, which are distal to the ABS, oligosaccharide modification of monoclonal antibodies was used to prepare conjugates. Conjugates modified with .sup.131 I coupled to a tyrosine-containing peptide, which was then site-specifically attached to oxidized oligosaccharides, exhibited greater targeting efficiency compared to the conjugates that were modified nonselectively on tyrosine. Because the use of Asn-297-associated carbohydrate requires the presence of the Fc portion of the antibody, its use is limited. There are certain applications employing antibody fragments in which the Fc portion is not present.